Team:SEFLS Shanghai/Improve

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Improve

Improve

Our Improvement

In this study, in order to improve the supply of precursor IPP and DMAPP, we tried to optimize the MEP pathway and explored the function optimization of gene ispG in the MEP pathway. IspG is included in the iGEM registry of standard biological parts, but only has some simple information. So we further studied the role of IspG in the MEP pathway, and constructed composite parts and other genes leading to a more efficient MEP pathway, which further improved the supply of precursor IPP and DMAPP.

It was demonstrated that balanced activation of IspG and IspH could improve isoprenoids production. To get an appropriate ratio between ispG and ispH gene expression strength， ispG expression strength can be incresed to medium level and ispH expression strength to high level.

Therefore, during the design, by placing the gene ispG on pBBR1MCS-2-Dxs-IspG (pMEP-DG) with a low-copy skeleton containing medium lac promoter to reduce its expression intensity, we constructed pbbr1mcs-2-Dxs-IspG (pMEP-DG). Gene Dxs was additionally introduced to be a key speed limiting enzyme in the MEP pathway. Placing gene ispH on a high-copy skeleton pETDuet-1 containing strong promoter T7 to increase its expression intensity, building pETDuet-1-1T7-IspH-Idi-IspA-Yss (pET-HIAY) and adding genes Idi and IspA are all ways to improve the supply of squalene synthetic precursor FPP.

We tried to replace the chassis cell, BL21(DE3), with XL1-blue. BL21(DE3) was suitable for expression vector containing T7 promoter (such as pET series), while the chassis was replaced with XL1-blue. We needed to construct pUC-HIAY by constructing gene ispH on pUC19m, a high-copy skeleton vector with lac promoter.

P35151, pMEP-DG and pUC-IAY were combined to co-transform E. coli XL1-blue and construct strain XH3, whose squalene production was 293.7mg/L.

When p35151, pMEP-DG and pUC-HIAY were combined to construct strain XH4, the squalene yield was 504.0mg/L, 1.7 times higher than that of strain XH3, indicating that the combinations of IspG and IspH with different overexpression intensity could be applied to different E. coli chassis cells to optimize the MEP pathway and improve the supply of precursor IPP and DMAPP in the chassis cells.